Abstract LB134: Type 1 calreticulin mutations differentially activate the IRE1α-XBP1 pathway of the unfolded protein response to drive myeloproliferative neoplasms
Authors: Juan Ibarra, Yassmin Elbanna, Katarzyna Kurylowicz, Michele Ciboddo, Harrison S. Greenbaum, Nicole S. Arellano, Deborah Rodriguez, Maria Evers, Dongbo Yang, Althea Bock-Hughes, Chenyu Liu, Quinn Smith, Julian Baumeister, Milena Kalmer, Kathrin Olschok, Benjamin Nicholson, Diane Silva, Jonathan Dowgielewicz3, Elisa Rumi, Daniela Pietra, Ilaria Carola Casetti, Steffen Koschmieder5, Sandeep Gurbuxani, Rebekka K. Schneider, Scott A. Oakes, Shannon E. Elf
Journal: Cancer Research (2022)

Abstract Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). 80% of CALR mutations are classified as either type 1 or type 2, exemplified by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5), respectively. Despite their shared mutant C-termini and mutual ability to bind and activate MPL, patients with type 1 and type 2 CALR mutations display significant clinical and prognostic differences. Type 1 mutations are primarily associated with an MF phenotype and a higher risk of fibrotic transformation from ET, while type 2 mutations are more common in ET. Molecularly, type 2 CALR mutant proteins retain many of the calcium binding sites present in the wild type protein, while type 1 CALR mutant proteins lose these residues. The functional consequences of this differential loss of calcium binding sites remain yet unexplored. Current targeted therapies for CALR mutated MPN are not curative, and treatment does not differentiate between type 1 versus type 2 mutant CALR-driven disease, despite the different phenotypic and prognostic outcomes in these patients. In order to improve treatment strategies for CALR mutated MPN patients, it is critical to identify specific dependencies unique to each CALR mutation type that can be exploited for therapeutic gain. Here, we show that type 1 CALRdel52 but not type 2 CALRins5 mutations lead to activation of and dependency on the IRE1α-XBP1 pathway of the unfolded protein response (UPR). Mechanistically, we found that the loss of calcium binding residues in the type 1 mutant CALR protein directly impairs its calcium binding ability, which in turn leads to depleted ER calcium and subsequent activation of the IRE1α-XBP1 pathway. Using cell lines and primary MPN patient samples, we identified two novel transcriptional targets of XBP1 specific to CALRdel52-expressing cells - the anti-apoptotic protein BCL-2 and the calcium efflux channel IP3R. We show that BCL-2 acts downstream of XBP1 to promote survival in the face of depleted ER calcium, while IP3R is up-regulated downstream of XBP1 to promote continued ER calcium efflux in order to sustain IRE1α-XBP1 pathway activation and survival. We found that genetic or pharmacological inhibition of IRE1α-XBP1 signaling induced cell death only in type 1 mutant but not type 2 mutant or wild type CALR-expressing cells. Moreover, we show that in vivo inhibition of IRE1α significantly abrogates type 1 mutant CALR-driven disease in a bone marrow transplantation model. This work is the first to demonstrate that type 1 and type 2 mutant CALR-expressing cells display differential molecular dependencies that can be exploited for therapeutic gain. Moreover, this study answers an enduring question regarding the functional consequence of the loss of calcium binding sites on the type 1 mutant CALR protein, and demonstrates how type 1 CALR mutant-expressing cells rewire the UPR, downstream calcium signaling, and apoptotic pathways to drive MPN. Citation Format: Juan Ibarra, Yassmin Elbanna, Katarzyna Kurylowicz, Michele Ciboddo, Harrison S. Greenbaum, Nicole S. Arellano, Deborah Rodriguez, Maria Evers, Dongbo Yang, Althea Bock-Hughes, Chenyu Liu, Quinn Smith, Julian Baumeister, Milena Kalmer, Kathrin Olschok, Benjamin Nicholson, Diane Silva, Jonathan Dowgielewicz3, Elisa Rumi, Daniela Pietra, Ilaria Carola Casetti, Steffen Koschmieder5, Sandeep Gurbuxani, Rebekka K. Schneider, Scott A. Oakes, Shannon E. Elf. Type 1 calreticulin mutations differentially activate the IRE1α-XBP1 pathway of the unfolded protein response to drive myeloproliferative neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB134.